1. Field of the Invention
The invention relates to an atomic force microscope which is operated at cryogenic temperatures under approximately ambient pressure, and more particularly, to the surface structural determination of biological and material science specimens, using a freeze-fracture/freeze-etch apparatus for surface structural determinations.
2. Brief Description of the Prior Art
The atomic force microscope (AFM), also called a scanning force microscope (SFM), unlike the scanning tunneling microscope, does not require the specimen to be either electron or ion conductive. Therefore, the atomic force microscope has applications in the biological sciences, as well as in the materials sciences. The high-resolution capability of the atomic force microscope is particularly attractive to researchers in structural biology, since crystallization is not required for atomic force microscope imaging.
Operating at room temperature in air, under vacuum, or in aqueous solutions, the atomic force microscope is capable of obtaining atomic resolution on hard crystalline specimens, like :mica and highly oriented pyrolytic graphite. However, for soft biological specimens, the resolution is much lower, generally in the range of nanometers. The atomic force microscope has been used to obtain images of various biological specimens, such as DNA, membrane proteins, and synthetic lipid and phospholipid supported bilayers. Resolutions limited to about 4-6 nm in air and 1-2 nm in buffer solutions have been achieved on substrate supported specimens in only a few cases.
The atomic force microscope uses a sharp stylus tip mounted on the end of a soft cantilever to probe the specimen surface. The interaction force between the tip and the specimen is responsible for the image contrast. Although the operation principle of such an instrument is simple, such a direct point contact approach to obtain images is fundamentally different from all other imaging instruments, such as electron or light microscopes, X-ray diffraction, and NMR. Since the contrast is obtained exclusively by localized interactions without any averaging, it poses stringent requirements on the shape and material of the tip, the mechanical strength of the specimen, and the adhesion between the specimen and the substrate. For biological applications of the atomic force microscope, the major limitation is the softness of the specimens, which in most cases prevents achieving very high spatial resolution. Further details are found in Mou et al., An Optical Detection Low Temperature Atomic Force Microscope at Ambient Pressure for Biological Research, Rev. Sci. Instrum. 64 (6), June 1993. The subject matter of the article is incorporated herein by reference, as though recited in full.
Attempts to improve the atomic force microscope have centered around specimen preparation and tip manufacture. While the use of sharpened tip did :not yield much improvement in the spatial resolution, the use of supported specimens provided improved resolution, but below the level which is required to resolve an alpha-helix of a protein. The use of a sharper tip requires the exertion of a larger local pressure, thereby exacerbating the problem of specimen deformation and/or damage. Severe deformation is already apparent with currently available tips at sub-nN force.
The imaging of bio-specimens at low temperature would appear to be one approach to improving the resolution of atomic force microscopes on biomacromolecul. es to the sub-nm range, since at the temperature of liquid nitrogen or similar low temperature liquid, most bio-materials showed a dramatic increase in mechanical strength (Young's modulus increased by a factor of 10.sup.3 to 10.sup.4). When combined with the modification of the well established techniques of freeze-fracture and freeze-etch, the ice matrix can also serve as a convenient solid substrate support. However, a major disadvantage of this approach is that surface contamination must be eliminated before this approach can be useful. The contamination problem is much more severe at cryogenic temperatures, because most of the contaminants will condense on the specimen surface, obscuring the features to be studied. A layer of such condensation would most likely appear as a nonuniform surface coverage due to the lack of control, and thereby prevent the imaging of specimen surface topology by the atomic force microscope.
An ultra-high vacuum (UHV) system may reduce such condensation to some extent. However, although the cryogenic pump becomes extremely efficient at 4.2 K, it would be difficult to reach the required vacuum at higher temperatures on the order of 77 K. Moreover, the maintenance of an ultra-high vacuum environment becomes more difficult and expensive, if not impossible, due to the presence of water in most preparations of biological specimens and an ice matrix as a solid substrate support because some etching will unquestionably occur. At intermediate vacuum (10.sup.-6 torr), such as the level used in most freeze-fracture/freeze-etch apparatus for replica making in electron microscopy, it has been shown that a complete surface coverage of contaminants takes about 30 minutes. In fact, a low-vacuum, low-temperature atomic force microscope system has been reported which shows that severe surface contamination preventing atomic resolution imaging of mica, occurred even before the temperature of 150 K was reached. The disadvantage of using UHV systems in the atomic force microscopy of material science specimens is removing volatile specimen components, such as O.sub.2 dopents in superconductors, by evaporation.